CN1285402A

CN1285402A - Process for separation of lysozyme

Info

Publication number: CN1285402A
Application number: CN 00128951
Authority: CN
Inventors: 文章军; 钱生球
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2000-09-18
Filing date: 2000-09-18
Publication date: 2001-02-28
Anticipated expiration: 2020-09-18
Also published as: CN1108381C

Abstract

The method for separating lysozyme includes the following steps: diluting egg albumin as raw material with proper quantity of water, stirring uniformly, adding acid to regulate pH value to 4.0-5.0, standing still, filteirng and removing deposit matter, heating filtrate to 75-80 deg.C, cooling to room temp., standing still for above 10 hr., filtering to obtain supernatant fluid, adding alkali to regulate pH value to 7.5-8.0, adding ethyl alcohol until white precipitate is produced, filtering, washing and drying so as to obtain the invented product. Said invented production process is simple, short in production period, high in yield, and said invented product can be used as food additive proservative.

Description

A kind of method of separating N,O-Diacetylmuramidase

The present invention relates to N,O-Diacetylmuramidase separation method technical field.

Corrupt for preventing food because of contaminating microorganisms, adopt the sanitas that adds chemosynthesis to restrain bacterial multiplication usually to prolong the storage life of food.The chemical substance of these interpolations, great majority have certain toxicity or side effect to human body.Seeking the Nantural non-toxic sanitas, is the target that the scientific worker makes joint efforts for a long time.

" Food science " (o. 11th 37-40 page or leaf in 1994) points out that N,O-Diacetylmuramidase is a kind of natural protein, and it can dissolve the cell walls of many bacteriums, causes bacteriolysis, has both had anti-corrosion function, again human body is had no side effect, and has very big using value." Food science " (the 12nd phase of nineteen ninety-five 6-9 page or leaf) report, lysozyme is than horn of plenty in the egg white, and its content accounts for the 3.5-4% of egg white, and about 96% protein does not have the bacteriolyze activity and becomes foreign protein in addition.

According to U.S.'s " Food science magazine " (Journal of Food Science, 1986 the 4th phase 1032-1036 pages or leaves), " Food science " (the 12nd phase of a nineteen ninety-five 6-9 page or leaf) and day clear 61-134323 of disclosure special permission communique report that the separation method of existing N,O-Diacetylmuramidase has affinity chromatography, ion exchange method, the precipitator method and precipitation and chromatography combined techniques.Affinity chromatography and ion exchange method all need be through to adorning the operating process such as dialysis, freeze-drying, carrier cleaning and regeneration of saltouing of post, the absorption of raw material upper prop, damping fluid washing, elutriant wash-out, product after the carrier pre-treatment, step is many, the cycle is long, the N,O-Diacetylmuramidase yield is low, only is 40-45%; There are the shortcoming that step is many, the cycle is long too in the precipitator method and precipitation and chromatography combined techniques.

The objective of the invention is to propose that a kind of processing step is brief, yield is high, cost is low, operation is the N,O-Diacetylmuramidase separation method of row easily, to overcome the above-mentioned shortcoming of prior art.

The present invention separates the method for N,O-Diacetylmuramidase, it is characterized in that: with the Ovum Gallus domesticus album is raw material, adds water suitably after the dilution, stirs, and the acid that adds weight concentration and be 5-10% transfers pH value to 4.0-5.0, leaves standstill, and removes by filter precipitation; Filtrate is warming up to 75-80 ℃, is cooled to room temperature, leave standstill and be no less than 10 hours, filter supernatant liquor, the alkaline solution that adds weight concentration and be 1-5% transfers pH value to 7.5-8.0, adds ethanol again to producing white precipitate, leaves standstill 6-10 hour; Filter, will precipitate, be drying to obtain product with the dehydrated alcohol flushing.

The described water that adds in Ovum Gallus domesticus album suitably dilutes, and is in order to reduce the viscosity of protein water soln, makes the foreign protein sedimentation after helping transferring pH value or heating, thereby reaches the purpose that foreign protein separates with N,O-Diacetylmuramidase.

It is food grade hydrochloric acid, citric acid or the lactic acid of 5-10% that weight concentration is selected in the acid that described accent pH value is used for use; It is food grade NaOH, the Na of 1-5% that the alkali that described accent pH value is used is selected weight concentration for use ₂CO ₃Or NaHCO ₃The aqueous solution.The concentration of acid, alkaline solution too conference is destroyed the protein activity of lysozyme.

By detecting as can be known, protein in the egg white, except that N,O-Diacetylmuramidase, other isoelectric point of protein is mostly between PH4.0-5.0, and therefore the iso-electric point of N,O-Diacetylmuramidase is up to PH10.7-11.0,, the present invention has adopted the isoelectric precipitation method, add acid and transfer pH value to 4.0-5.0, most of foreign protein precipitation is removed, then remaining protein is mainly N,O-Diacetylmuramidase in the solution; Because N,O-Diacetylmuramidase has thermotolerance, under acidic conditions, can stand the pyroprocessing of long period and non-loss of activity, foreign protein then will sex change when being heated to 75 ℃ and is precipitated, therefore the inventive method has adopted and has heated lysozyme soln in short-term and make foreign protein sex change precipitation to 75-80 ℃: after the cooling, available certain density alcohol replaces water and N,O-Diacetylmuramidase forms hydrogen bond, reduce the solvability of N,O-Diacetylmuramidase, thus precipitable N,O-Diacetylmuramidase.Adopt the inventive method to divide exsolution mattress enzyme, whole process can be finished at room temperature 24 hours, and processing step is brief, operation is simple; Raw materials consumption is few, the recyclable utilization of foreign protein, and N,O-Diacetylmuramidase yield height, cost is low: the N,O-Diacetylmuramidase of the gained crystal that is creamy white, it is sweet to distinguish the flavor of, and is applicable to the foodstuff additive of anticorrosion usefulness, and food is had no adverse reaction.

The N,O-Diacetylmuramidase that adopts the inventive method gained is added fresh milk by a certain percentage and is coated on the steamed bun, compare test with fresh milk that does not use N,O-Diacetylmuramidase and steamed bun, the result proves with the made N,O-Diacetylmuramidase of present method to have good preservative activity.

The inventive method and conventional ion exchange resin method compare:

Method	Production stage	Production cycle (hour)	Per kilogram egg white lysozyme (gram)
Ion exchange method	7 steps	56	1.6
The inventive method	3 steps	24	3.0

This shows, adopt the inventive method to separate N,O-Diacetylmuramidase, processing step letter, with short production cycle, N,O-Diacetylmuramidase yield height; Prepared N,O-Diacetylmuramidase can be used as the foodstuff additive of anticorrosion usefulness, favorable anti-corrosion effect.

Below be embodiments of the invention.

Embodiment 1:

Get 1.5 kilograms in freshly-slaughtered poultry egg white, add 3.0 kilograms of dilutions of deionized water to reduce viscosity, after stirring, the adding weight concentration is 10% edible hydrochloric acid accent pH value to 4.0, and this moment, the adularescent precipitation produced, and stopped to add acid, left standstill 1 hour, removed by filter precipitation; Filtrate moved to be heated to 75 ℃ in the water-bath, kept 3 minutes, take off the container of dress filtrate, drench in container outside with tap water and to be chilled to room temperature, leave standstill 10 hours after, use filter paper filtering, taking out supernatant liquor, is that 4% the NaOH aqueous solution is transferred pH value to 7.5 with filtrate with weight concentration, adds volumetric concentration and is 95% food grade ethanol and precipitate to producing, leave standstill after 8 hours and use filter paper filtering, to precipitate with the dehydrated alcohol flushing, dry under the vacuum, the crystal that obtains being creamy white, it is sweet to distinguish the flavor of, and is the product N,O-Diacetylmuramidase.

25 ± 3 ℃ of room temperatures, under the experimental situation of medial humidity 46%, in the 100ml fresh milk, add 0.2 gram N,O-Diacetylmuramidase, stir and make its dissolving, in the 100ml fresh milk, do not add anything in addition and compare, observe its appearance change every day; Under same environmental conditions, to get 0.2 gram in the ratio of 0.1% (with the weight ratio of steamed bun) and be dissolved in the 10ml deionized water for stirring and become pasty state to coat on a slice steamed bun, another sheet does not add anything and compares, and observes its appearance change every day.The result is as follows:

Table 1: the anticorrosion contrast experiment of milk:

(-) expression no change in the last table, (+) expression has peculiar smell, (++) expression upper strata virescence, (+++) layering of expression solution.

Table 2: the anticorrosion contrast experiment of wheaten food steamed bun:

(-) expression no change in the last table, (+) represents that the flavor of turning sour is arranged, mildew appears in (++) expression, (+++) the large stretch of mildew of expression appearance.

Embodiment 2

Get 1.5 kilograms in freshly-slaughtered poultry egg white, add 2.0 kilograms of deionized waters, after stirring, the adding weight concentration is 6% edible citric acid accent pH value to 4.5, and this moment, the adularescent precipitation produced, and stopped to add acid, left standstill 2 hours, and removed by filter precipitation, filtrate is moved to be heated to 75 ℃ in the water-bath, kept 3 minutes, take off container, drench in container outside with tap water and be chilled to room temperature, leave standstill 12 hours after, taking out supernatant liquor with filter paper filtering, is 5% NaHCO with weight concentration ₃The aqueous solution transfers to PH 8.0 with filtrate, and the adding volumetric concentration is 95% extremely generation precipitation of food grade ethanol, leaves standstill after 10 hours and uses filter paper filtering, will precipitate with the dehydrated alcohol flushing, is drying to obtain the finished product N,O-Diacetylmuramidase under the vacuum.

Adopt the mode identical with embodiment 1 to make anticorrosion simultaneous test the N,O-Diacetylmuramidase of present embodiment gained, the result is identical with embodiment's 1.

Embodiment 3

Get 1.5 kilograms in freshly-slaughtered poultry egg white, add 3.0 kilograms of deionized waters, after stirring, the adding weight concentration is 10% edible lactic acid accent pH value to 5.0, and this moment, the adularescent precipitation produced, and stopped to add acid, left standstill 1.5 hours, and removed by filter precipitation, filtrate is moved to be heated to 80 ℃ in the water-bath, kept 3 minutes, take off container, drench in container outside with tap water and be chilled to room temperature, leave standstill 10 hours after, taking out supernatant liquor with filter paper filtering, is 2% Na with weight concentration ₂CO ₃The aqueous solution is transferred pH value to 8.0 with filtrate, and the adding volumetric concentration is 95% extremely generation precipitation of food grade ethanol, leaves standstill after 7 hours and uses filter paper filtering, and will precipitate with the dehydrated alcohol flushing, is drying to obtain the finished product N,O-Diacetylmuramidase under the vacuum.

Claims

1, a kind of method of separating N,O-Diacetylmuramidase is characterized in that: be raw material with the Ovum Gallus domesticus album, add water suitably after the dilution, stir; The adding weight concentration is that the acid of 5-10% transfers pH value to 4.0-5.0, leaves standstill, and removes by filter precipitation; Filtrate is warming up to 75-80 ℃, is cooled to room temperature, leave standstill and be no less than 10 hours; Filter and take out supernatant liquor, the adding weight concentration is that the alkaline solution accent pH value of 1-5% is 7.5-8.0; Add ethanol to producing white precipitate, left standstill 6-10 hour, filter, will precipitate, be drying to obtain product with the dehydrated alcohol flushing.

2, separate the method for N,O-Diacetylmuramidase according to claim 1, it is characterized in that: it is food grade hydrochloric acid, citric acid or the lactic acid of 5-10% that weight concentration is selected in the acid that described accent pH value is used for use.

3, separate the method for N,O-Diacetylmuramidase according to claim 1, it is characterized in that: it is food grade NaOH, the Na of 1-5% that the alkaline solution that described accent pH value is used is selected weight concentration for use ₂CO ₃Or NaHCO ₃The aqueous solution.